Significance of NatB-mediated N-terminal acetylation of auxin biosynthetic enzymes in maintaining auxin homeostasis in Arabidopsis thaliana

The auxin IAA (Indole-3-acetic acid) plays key roles in regulating plant growth and development, which depends on an intricate homeostasis that is determined by the balance between its biosynthesis, metabolism and transport. YUC flavin monooxygenases catalyze the rate-limiting step of auxin biosynthesis via IPyA (indole pyruvic acid) and are critical targets in regulating auxin homeostasis. Despite of numerous reports on the transcriptional regulation of YUC genes, little is known about those at the post-translational protein level. Here, we show that loss of function of CKRC3/TCU2, the auxiliary subunit (Naa25) of Arabidopsis NatB, and/or of its catalytic subunit (Naa20), NBC, led to auxin-deficiency in plants. Experimental evidences show that CKRC3/TCU2 can interact with NBC to form a NatB complex, catalyzing the N-terminal acetylation (NTA) of YUC proteins for their intracellular stability to maintain normal auxin homeostasis in plants. Hence, our findings provide significantly new insight into the link between protein NTA and auxin biosynthesis in plants.

During plant growth and development and response to environmental stimuli, auxin biosynthesis is regulated at multiple levels, including epigenetic regulation 14 , transcriptional initiation 12,15 , and post-translational protein modifications 16 . Among diverse post-translational modifications, N-terminal acetylation (NTA) is a highly abundant protein modification in eukaryotes in which the acetyl group of acetyl-CoA is transferred to the amino group of a protein at its N-terminus under the catalysis of N-terminal acetyltransferases (NATs) 17,18 , altering the steric or chemical properties of the modified N-terminus that may affect protein-protein interaction 19,20 , subcellular localization 21,22 , protein folding and aggregation 23,24 , stability or degradation [25][26][27] in normal cellular life in growth, development, and responses.
Although universally prevalent in eukaryotes, the functional significance of NTA has only recently emerged in plants, with a limited number of reports on its roles in growth, flowering, reproduction 28,29 , stress 30 and immunity 27 . However, functional significance of NTA in auxin biosynthesis has not been reported so far. Here, we show evidences that the Arabidopsis NatB catalyzed NTA of YUC proteins can stabilize their intracellular stability to maintain normal auxin homeostasis in plants. Hence, our findings provide significant new insight into the link between protein NTA and auxin biosynthesis in plants.
Results and discussion ckrc3 is an auxin-deficient mutant. Auxin homeostasis in plants is crucial to control many aspects of plant growth and development 1,31 . To study its regulation, novel auxin-deficient mutants are vital for uncovering the unknown genes functioning in auxin homeostasis. However, the complexity of the auxin biosynthesis and metabolic pathways has greatly hindered the efforts to dissect their molecular mechanisms. To overcome this problem, a large-scale screening for auxin-deficient mutants has previously performed in our lab, in which the ckrc3 mutant was isolated as one of the so-called group II cytokinin (CK) induced root curling (ckrc) mutants 32 . When grown on medium containing 0.1 μM trans-zeatin (tZ), these mutants displayed a root curling phenotype, which is usually caused by auxin deficiency 11 . Besides some general pleiotropic developmental defects, such as the significantly reduced growth rate ( Fig.1f-g; Supplementary  Fig. 1d, i), leaf reticulation, early flowering, aborted ovules in short silique ( Supplementary Fig. 1b, c, f, h 33 ), impaired apical hooks ( Supplementary Fig. 1e, g), ckrc3 mutant also exhibited typical auxin-deficient phenotypes in roots, including the reduced length of primary roots, wavy or CK-induced curling growth, defective gravitropic response (Fig. 1a-e), less number of root hairs ( Supplementary Fig. 1a), weaker expression of the auxin reporters DR5::GUS or DR5::GFP (Fig. 1h, i) and reduced endogenous contents of free IAA and its metabolites (IAGlu, IAAsp, and especially oxIAA) 32 , which can be rescued either by exogenous application or endogenous production (by superroot2, sur2 mutation) of IAA (Fig. 1a, c, d, j; Supplementary Fig. 2). Previously it was known that the defects in glucosinolate biosynthesis by the sur2 mutation can lead to auxin accumulation 34,35 , which can be used to rescue auxin deficiency 8 .
CKRC3 gene encodes the auxiliary subunit which interacts with NBC to form a NatB complex for maintaining endogenous auxin levels. Genetic analyses and map-based cloning identified ckrc3 as a loss of function mutation in the AT5G58450 gene. The mutation caused a premature termination by a G -> A transition, changing the tryptophan (at the 731th aa. position of the encoded protein) codon TGG into the TAG stop codon (Supplementary Fig. 3a-d). Genetic allelic analyses and molecular complementation confirmed that AT5G58450 is the CKRC3 gene (Supplementary Fig. 3e-g).
CKRC3 is annotated to encode the auxiliary subunit (Naa25) of a putative NatB in Arabidopsis, and was named TCU2 previously 33 . NatB is one of the eight NATs, NatA through NatH found so far in eukaryotes 36,37 , specifically catalyzing the acetylation of proteins beginning with iM-D/E/N/Q [38][39][40] .
The Arabidopsis NatB is presumed to contains a catalytic subunit Naa20 (NatB catalytic subunit, NBC) and an auxiliary subunit Naa25 (CKRC3/TCU2) 33 . In Candida albicans, Naa25 forms a horse-shoe-like deck to hold specifically its catalytic subunit Naa20 40 . The Arabidopsis Naa25/CKRC3/TCU2 protein has 1065 amino acids and contains several tetratricopeptide repetitions (TPRs) in its N-terminal part. TPR domains are composed of 3-16 degenerate tandem repeats of 34 amino acids that form structural domains in proteins that facilitate the assembly of large protein complexes 41,42 . CKRC3/TCU2 also contains a NatB domain with unknown function in the intermediate region, which is very conserved and occupies half of the NatB auxiliary subunit in all organisms studied ( Supplementary Fig. 4a). As predicted, a strong interaction between CKRC3/TCU2 and NBC was detected by both yeast twohybrid (Y2H) (Fig. 2a) and Co-IP assays in tobacco cells ( Fig. 2b; Supplementary Fig. 14), which depends on both the TPR motif and NatB domain, as shown by experiments with different truncations of CKRC3/TCU2 ( Supplementary Fig. 4), illustrating that CKRC3 and NBC can interact to form NatB complexes both exo and in planta, as also reported elsewhere 43 after the initial submission of this work. These results are consistent with the cytoplasmic co-localization of CKRC3/TCU2 and NBC, as revealed by CKRC3/TCU2-eGFP and NBC-GFP fusion proteins ( Supplementary Fig. 5), and the similar auxin-deficient phenotypes between ckrc3, nbc-1 and ckrc3 nbc-1 (  Supplementary Fig. 6). Hence, the interaction between NAA25/CKRC3/TCU2 and NAA20/NBC to form a NatB complex is required for maintaining normal IAA levels in Arabidopsis.
NatB complex catalyzes N-terminal acetylation of YUC8 for its intracellular stability to maintain auxin homeostasis. Among the known auxin biosynthetic pathways, YUCs catalyze the ratelimiting step of auxin biosynthesis via IPyA and are critical targets in regulating auxin homeostasis 44,45 . In Arabidopsis, 8 of the 11 YUCs 46 possess NatB substrate signatures (ME…, Supplementary Fig. 7). Among YUCs, which are highly functional redundant 46 , only the loss of function mutant of YUCCA8 (yuc8) had ckrc phenotype ( Supplementary Fig. 8). Because of this phenotypic advantage, we focused YUCCA8 (YUC8) in further investigations.
To confirm that YUC8 is a target protein of NatB, we first examined the enzyme activity of NBC in vitro. We used synthesized oligo-polypeptides as the substrates. NTA modification was detected by LC-MS/MS and found to occur on both the positive control N-terminal peptide of SNC1 protein 27 and that of our YUC8(iME-) target, but not in the negative control of the mutated form of YUC8(iMAE-), in which an insertion of A between M and E could theoretically disrupt the NatB substrate signature (Supplementary Fig. 9; Supplementary Tab. 1). The in vitro NTA of the potential N-terminal peptide substrates of other YUCs by NatB were also confirmed (Supplementary Tab. 1).
To detect the in vivo NatB-mediated NTA, we expressed YUC8 N7 -YFP fusion protein in transgenic plants. Ac iMet were detected in YUC8 N7 -YFP by LC-MS/MS in the yuc8 single mutant but not in yuc8 ckrc3 double mutant background ( Supplementary Fig. 12), confirming that the NTA of YUC8 were mediated by NatB complex.
To investigate the role of NatB-mediated NTA of YUC8 in auxin homeostasis in plants, we generated CaMV 35S::YUC8-mGFP transgenic lines in both wild-type (WT) (#1 and #13) and ckrc3 nbc-1 (#3 and #7) backgrounds, and over-expression of YUC8-mGFP was obtained in all of these 4 independent transgenic lines (Fig. 3c), which produced high auxin phenotypes (long hypocotyl and epinastic cotyledons) in WT (Fig. 3a, b), indicating that the fusion protein is fully functional; but not in the ckrc3 nbc-1 double mutant (Fig. 3a, b), revealing that NatBmediated NTA of YUC8 is crucial for its function in auxin biosynthesis, most likely by promoting the stability of YUC8 protein, as reported elsewhere for SNC1 protein 27 , which was confirmed in our present study by transient expression of YUC8-eGFP fusion protein in Arabidopsis mesophyll protoplasts ( Supplementary Fig. 10) and WB analysis on protein abundance in transgenic plants ( Fig. 3d; Supplementary Fig. 15), where the transcript levels was comparable between WT and the ckrc3 nbc-1 double mutant (Supplementary Fig. 13).
The substrate specificities of NATs are generally determined by the first two amino acids of the target protein 47 , consequently, the substitution of the second residue would principally modulate the NTA, influencing the stability and/or biological function of the target protein. To further test the critical Nt-residues and the substrate specificity of NatB-mediated NTA of YUC8 in plants, we substituted the second E by A, a small aliphatic residue, generating a mutant form of YUC8(E2A). In vitro assay showed that the E2A substitution did not significantly alter the enzyme activity of YUC8 (Supplementary Figs. 11;17), but greatly reduced the protein abundance in its over-expression plants (Fig. 4d, e; Supplementary Fig. 16) when compared with wildtype YUC8 ME -mGFP), causing a failure either to produce high auxin phenotypes, or to rescue the yuc8 root phenotypes (Fig. 4a-c). These results again reveal that the intracellular stability of YUC8 for its steady-state level can largely depend on Data are presented as mean ± SD, ** P < 0.01 according to ANOVA followed by Tukey's multiple comparison tests. e Quantification of root gravitropic responses (n ≥ 50). f, g Rosettes and fresh weight of 30 DAG plant (n = 31). Data are presented as mean ± SD, *** P < 0.001 according to ANOVA followed by Tukey's multiple comparison tests. h, i GUS staining or GFP fluorescence of DR5::GUS/GFP marker lines, bar = 100 μm. j Primary root length at 7 DAG on MS medium with increasing concentrations of IAA (n ≥ 15). Data are presented as mean ± SD.
the NatB activity. During the revision of this paper, Li et al (2020) 48 reported similar NatB-mediated stabilization of the stress/immune-related SIGMA FACTOR-BINDING PROTEIN1 (SIB1) in Arabidopsis.

Conclusions
In summary, our present study reveals that in Arabidopsis NatB complex of CKRC3/TCU2-NBC can mediate the NTA of YUC8 for its intracellular stability to maintain auxin homeostasis (Fig. 4f) for normal growth. In Arabidopsis, 8 of the 11 YUCs possess NatB substrate signatures ( Supplementary Fig. 7), catalyzing the rate-limiting step of the main auxin biosynthesis pathway (IPyA pathway) in various tissue/organs 7,49 . As NatB is widely expressed in tissues throughout plant development (Supplementary Fig. 5a, c, d). NatB-mediated NTA of these YUCs must play significant roles in auxin homeostasis at the whole plant level for normal growth and development.

Methods
Plant material and growth conditions. Information about the Arabidopsis thaliana mutants used in this study listed in Supplementary Tab. 2. The mutants were confirmed by PCR and sequencing. The primers used for PCR are given in Supplementary Tab. 3. Double mutant lines were obtained by crossing. For construction of transgenic vector, the full-length CDS of YUC8 was amplified by RT-PCR from Col-0 and cloned into pCAMBIA1302-mGFP vector for generating Fig. 2 Evidences of interaction between CKRC3/TCU2 and NBC to form a NatB for maintaining endogenous auxin levels. a Y2H test in vitro, with pGBKT7-53 and pGADT7-T as positive, pGBKT7-lam and pGADT7-T as negative controls, respectively. b Interaction between CKRC3 and NBC in vivo revealed by Co-IP test after transiently co-expressing 35S::Myc-CKRC3 and 35S::HA-NBC in N.benthamiana leaves. The nuclear protein ICE1 was used as negative control. c-e Plant phenotypes at 7 DAG; bar = 5 mm, n ≥ 12. Data are presented as mean ± SD, different capital letters indicate significant differences at P < 0.01 according to ANOVA followed by Tukey's multiple comparison tests. f GUS staining of DR5::GUS marker lines, bar = 100 μm. g Quantification of root gravitropic responses (n ≥ 39). h, j Rosettes and fresh weight of 30 DAG plant (n = 31). Data are presented as mean ± SD, different capital letters indicate significant differences at P < 0.01 according to ANOVA followed by Tukey's multiple comparison tests. i Primary root length at 7 DAG on MS medium with increasing concentrations of IAA (n ≥ 15). Data are presented as mean ± SD.
Seeds were sterilized with 0.1% HgCl 2 , thoroughly washed with dH 2 O three times, and placed on Murashige Skoog (MS) medium containing 1% agar and 1% sucrose. Before being transferred to culture under long-day conditions (16 h light/ 8 h dark) at 22°C, the plates were kept at 4°C for 2 days in darkness. After 7 days of growth, the plants were analyzed phenotypically or transferred to soil and cultivated in a greenhouse at the same growth conditions as above.
Phenotype characterization. For root growth inhibition assays and biochemical complementation, seeds were germinated and grown vertically on MS medium with different hormones at a selected range of concentrations at 22°C with a 16/8 hour light/dark cycle for 7 days, then their root elongation was measured. All presented data were the mean values of three separate experiments using at least 60 seedlings for each replica.
The Degree of root Curling was calculated by dividing the distance between the two ends of a root (L 0 ) by its actual length (L).
For root gravitropic response assays, the germinated seedlings were first grown vertically on MS plates for 6 days at 22°C with a 16/8 hour light/dark cycle, then transferred to fresh media. After 3 hours, the plates were rotated 90 degrees and grown horizontally for 24 hours. The degree of gravitropic response was measured for each root. Approximately 100 seedlings were measured for each genotype and treatment.
Constructs for subcellular localization. The full-length coding sequences of CKRC3/TCU2 and NBC were amplified by PCR using their respective specific pair of primers, and cloned via recombination technology into the pCAMBIA2300 vector with the GFP coding sequence to construct pCAMBIA2300-eGFP and pCAMBIA2300-GFP vectors. GFP fusion proteins were expressed in tobacco cells 2 days after transient transformation. The GFP fluorescence was analyzed with a laser-scanning confocal microscope (Leica, https://www.leica.com/). RNA extraction and quantitative real-time PCR. RNA was isolated using Trizol (Invitrogen, http://www.invitro-gen.com/) and reverse-transcribed using a reverse transcription kit (Takara, http://www.takara-bio.com/). Quantitative RT-PCR was performed in CFX (Bio-Rad, https://www.bio-rad.com/) real-time PCR equipment by using the SYBR green chemistry (Takara). ACTIN8 was used as an internal control. Quantitative PCR analysis was performed with three different replicates for each biological sample. Three biological replicates were performed in each experiment.
Y2H assays. Y2H were performed according to the instructions provided with the Matchmaker LexA two-hybrid system (Clontech, http://www.takara-bio.com/). pGBKT7-CKRC3/TCU2 constructs were obtained by inserting full-length coding sequences of CKRC3/TCU2 into the EcoRI and SalI sites of the pGBKT7 plasmid, and pGADT7-NBC constructs by inserting that of NBC. Corresponding pairs of plasmids were transformed into yeast strain. Yeast transformants were then plated on minimal SD/-Leu/-Trp agar plates and incubated for 4 days at 30°C. The wellgrown colonies were plated onto minimal SD/-Leu/-Trp/-His/-Ade agar plates with or without X-α-Gal and AbA for interaction tests for staining.
Immunoprecipitation (IP). Full-length coding sequences of CKRC3/TCU2-Myc and NBC-HA were introduced into pCAMBIA1302-mGFP vector. IP was performed with transient expression of CKRC3/TCU2-Myc and NBC-HA constructs in N. benthamiana leaves, the protein ICE1 located in nucleus as a negative control. Leaves (0.3 g) were collected 24 h after infiltration of 4 × 10 8 cfu/mL, ground to a powder under liquid nitrogen, and resuspended in 2.0 mL of IP buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% Nonidet P-40, 5 mM DTT, and 1.5×Complete Protease Inhibitor (Roche, https://www.roche.com.cn/). The b Hypocotyl length at 7 DAG; n = 12. Data are presented as mean ± SD, different capital letters indicate significant differences at P < 0.01 according to ANOVA followed by Tukey's multiple comparison tests. c YUC8 expression levels determined by qRT-PCR. Data are presented as mean ± SD of three independent experiments, different capital letters indicate significant differences at P < 0.01 according to ANOVA followed by Tukey's multiple comparison tests. d YUC8-mGFP fusion protein levels determined by WB using anti-GFP antibody, ACTIN as internal reference.
Protein expression in prokaryotes and purification. Full-length coding sequence of NBC was introduced into pEASY-Blunt E2-His-MBP vector. The pEASY-Blunt E2-His-MBP-NBC plasmid was transformed into E. coli BL21 Star (DE3) cells (Invitrogen). Cell culture was grown in LB (lysogeny broth) medium to an OD 600nm of 0.6 at 37°C and subsequently transferred to 18°C. After 30 min of incubation, protein expression was induced by isopropyl-β-D-thiogalactoside (0.5 mM). After 12 h of incubation, the cultures were harvested by centrifugation (4000 rpm) at 4°C. E. coli pellets containing recombinant proteins were lysed by sonication in lysis buffer (1 mM DTT, 50 mM Tris-HCl (pH 7.4), 300 mM NaCl, 1 tablet EDTA-free protease Inhibitor cocktail per 50 ml (Roche). The cell extracts were applied on a Ni-TED 1 ml Sefinose (TM) Column (Sangon Biotech, https:// www.sangon.com/). The purity of the fractions corresponding to purified monomeric recombinant proteins was analyzed on Coomassie-stained SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels and the protein concentrations were determined by OD 280nm measurements.
In vitro NAT assay. Purified His-MBP-NBC (0.5 mM) was mixed with selected oligopeptide substrates (200 mM) and 300 mM of acetyl-CoA in a total volume of 60 μl acetylation buffer (50 mM Tris-HCl, pH 7.5, 10% glycerinum, 10 mM DTT, 1 mM EDTA). The samples were incubated at 37°C for 60 minute. The enzyme activities were quenched by adding 5 ml of 10% TFA. The acetylation reactions were quantified using RP-HPLC and LC-MS/MS. Synthetic peptides were custom-made to a purity of 90%. All peptides contain 10 unique amino acids at their N-terminus, as these are the major determinants influencing NTA. The next 17 amino acids are essentially identical to the ACTH peptide sequence (RWGRPVGRRRRPVRVYP) except that the lysines were replaced by arginines to minimize any potential interference by N-terminal acetylation. Peptide sequences: SNC1, MDTSKDDDMERW GRPVGRRRRPVRVYP; YUC8, MENMFRLMDQRWGRPVGRRRRPVRVYP; YUC8 (MAE), MAENMFRL MDQRWGRPVGRRRRPVRVYP.
Plant protein extraction. Plant tissue was ground in liquid nitrogen to a fine powder. The protein extraction buffer (50 mm Tris-HCl [pH 7.5], 150 mm NaCl, 5 mm DTT, 10% [v/v] glycerol, 1% [v/v] Nonidet P-40, and 1×Complete protease inhibitor cocktail) was added at a 1:3 ratio (tissue:buffer) to the powder and mixed well. The supernatant was harvested after two centrifugations at 13,000 rpm for 20 min in a 4°C table-top centrifuge. Then the protein content of all samples was quantified. The resulting proteins were resuspended in 2× SDS sample buffer and denatured for 10 min at 98°C.
In vivo NAT assay. IP Coupled with MS Analysis. Total proteins were isolated from transgenic plants overexpressing YUC8 N7 ::eYFP fusion protein using the protein extraction buffer described above. The protein extract was incubated with 25-35 µL of anti-GFPmAb-magnetic beads (Chromotek, https://www.chromotek.com/) for 4 h at 4°C with constant rotation. The beads were then washed three times with the IP buffer. After the last wash, 25 µL of the beads was eluted in 50 μL of 2× SDS protein sample buffer by incubating at 98°C for 10 min. The eluates were separated by 12% SDS-PAGE, and detected by immunoblot analyses using a mouse anti-GFP monoclonal antibody (1:5,000; Abmart, http://www.ab-mart.com.cn/, Catalog no. M20004). Cutting the target bands from IP into cubes (ca. 1 ×1 mm). The proteins were reduced, alkylated, and destained in gel, saturated, and digested by trypsin for LC-MS/MS (nanoLC-MS/MS, Orbitrap Fusion TM Lumos TM Tribrid TM ) analysis. The mass spectra were submitted to the Proteome Discoverer (Thermo Scientific, https://www.thermofisher.cn/cn/zh/home.html) for peptide identification. Fig. 4 NatB-mediated NTA of YUC8 is determined by N-terminal Aa and is essential for its intracellular stability to function in auxin biosynthesis. a Phenotypes of yuc8 mutant and transgenic yuc8 plants harboring YUC8/YUC8(E2A)-mGFP at 7 DAG on MS media with or without 0.1 μΜ tZ, bar = 5 mm. b, c Quantification of Hypocotyl length (MS, n ≥ 11) and degree of root curling (0.1 μΜ tZ, n ≥ 23). Data are presented as mean ± SD, different capital letters indicate significant differences at P < 0.01 according to ANOVA followed by Tukey's multiple comparison tests. d YUC8 expression levels determined by qRT-PCR. Data are presented as mean ± SD of three independent experiments, different capital letters indicate significant differences at P < 0.01 according to ANOVA followed by Tukey's multiple comparison tests. e YUC8-mGFP fusion protein levels determined by Western analysis using anti-GFP antibody, ACTIN as internal reference. f A proposed model elucidating the role of NatB in controlling the auxin biosynthesis.
SDS-PAGE and western blot. Protein samples were separated by SDS-PAGE and then transferred to a polyvinylidene fluoride film. After blocking with 5% bovine serum albumin, the film was incubated with primary antibody overnight at 4°C, then washed three times with PBST for 10 minute and incubated with secondary antibody for 1 hour at room temperature. After washing three times with PBST for 10 minute, the film was illuminated using a luminous imaging system. The YUC8-mGFP fusion protein was immunochemically detected using a mouse anti-GFP monoclonal antibody (1:2000; Abmart, Catalog no. M20004M). ACTIN protein was detected using mouse anti-Plant Actin monoclonal antibody (1:5000; Abbkine, https://www.abbkine.com/, Catalog no. ABL1050).
Transient expression in protoplasts. The transient expression was performed as described 51 . The YUC8-eGFP/YUC8 (E2A)-eGFP CDS was amplified by PCR and inserted into pCAMBIA2300 vector. Primers used are listed in Supplementary Tab. 3. For transient expression assay, Arabidopsis mesophyll protoplasts from the Col-7 and ckrc3 nbc-1 mutant were transfected with 200 μg plasmid and incubated 18 h. The GFP fluorescence was observed under a laser scanning confocal microscope Zeiss 880 (Zeiss, https://www.zeiss.com/corporate/int/home.html).
Statistics and reproducibility. All results are expressed as the means ± standard deviation. The numbers of samples and replicates of experiments were shown as mentioned in the figure legends. Comparisons between groups were determined using ANOVA followed by Tukey's multiple comparison test. All data were analyzed by using GraphPad Prism 8 software (https://www.graphpad.com/). Reporting summary. Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
All data generated during this study are included in the supplementary information files.